Method for editing bacillus subtilis genome based on CRISPR-Ascas12f system and application of method

Abstract

The invention discloses a method for editing a bacillus subtilis genome based on a CRISPR (clustered regularly interspaced short palindromic repeats)-Ascas12f system. The method comprises the following steps: step 1, constructing a CRISPR / Cas12f1 plasmid; step 2, editing a bacillus subtilis genome; and 3, verifying an editing result. According to the invention, the CRISPR-Cas12f technology is utilized to carry out rational transformation on the bacillus subtilis BS168 strain, and the yield of MK-7 is increased by knocking out the competitive pathway isochorismate lyase.

Description

Technical Field

[0001] The present invention relates to the technical field of Bacillus genome editing, and more specifically to a method for editing the Bacillus subtilis genome based on a CRISPR-Cas12f1 system and an application thereof. Background Art

[0002] Traditional strategies for genetic manipulation of Bacillus subtilis are based on homologous recombination. These genome editing methods include the use of antibiotic resistance markers, counter-selection markers, and the cre / loxP system. In recent years, the CRISPR-Cas9 system, which originated from the bacterial adaptive immune system, has been applied to the genetic manipulation of many prokaryotes and eukaryotes. The CRISPR-Cas9 system is highly efficient, time-saving to use, and the modified organisms are unmarked. Three different CRISPR-Cas9 systems have been used in Bacillus subtilis: a single-plasmid system, a double-plasmid system, and a chromosome system. With the assistance of a single guide RNA (sgRNA), the Cas9 protein can introduce double-strand breaks (DSBs) at the target site of the Bacillus subtilis genome. Then, the homologous repair template guides homologous recombination to modify, knock out, or insert genes. However, both CRISPR / Cpf1 and CRISPR / Cas9 have exposed defects and limitations, such as severe off-target effects, high toxicity, and low transformation efficiency, and are not suitable for industrial Bacillus subtilis with low transformation efficiency. Therefore, it is increasingly necessary to establish efficient and low-toxic CRISPR / Cas Bacillus subtilis genome editing technology to provide reliable operating tools for the genetic transformation of Bacillus subtilis with low DNA transformation efficiency.

[0003] Through the CRISPR / Cas12f system, a Bacillus subtilis strain that efficiently synthesizes MK7 was constructed to achieve efficient gene editing. Vitamin K2 is a general term for a class of compounds containing a 2-methyl-(1,4)-alkyne nucleus at the C3 position of the isoprene unit side chain. Vitamin K2 has 14 different compounds, and the number of isoprene units in the side chain structure is different in form. MK-7 is an important form among them and is also an indispensable nutrient in people's daily diet. At present, the MK-7 derived from food fermentation has a low content and cannot meet the needs of the human body. The chemical synthesis of vitamin K2 requires strict catalytic conditions, many by-products, and is expensive; therefore, large-scale production is a problem. The synthesis of MK-7 by microbial fermentation can reduce costs and pollution, and there is hope for industrialization. Isochorismate synthase catalyzes the first step of the MK-7 synthesis pathway. In B. subtilis, there are two genes encoding isochorismate synthase: menF and dhbC. DhbC and four other genes form the dhbA-dhbC-dhbE-dhbB-dhbF operon, which is used to synthesize 2,3-dihydroxybenzoic acid (DHB) and ultimately synthesize a low molecular weight iron ion chelator-enterobactin. The isochorismate lyase encoded by dhbB catalyzes the first step of DHB synthesis, that is, catalyzing the cleavage of isochorismate into 2,3-dihydroxy-2,3-dihydroxybenzoic acid (DHDHB). By knocking out the synthesis of 2,3-dihydroxybenzoic acid (DHB) in the competitive pathway, the production of MK7 can be effectively increased. Summary of the invention

[0004] The object of the present invention is to provide a method for editing the genome of Bacillus subtilis based on the CRISPR-Ascas12f system and its application.

[0005] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a method for editing the genome of Bacillus subtilis based on the CRISPR-Ascas12f system, comprising the following steps:

[0006] Step 1: Construction of CRISPR / Cas12f1 plasmid

[0007] Using pjoe8999 plasmid as template, reverse PCR was performed to obtain a backbone plasmid retaining the replicon and terminator; and pJOE-cas12f1 was formed by seamless cloning.

[0008] sgRNA-F and sgRNA-R were annealed and connected to pJOE-cas12f1 via GoldenGate to obtain pJOE-sgRNA12-cas12f1 plasmid;

[0009] Primers are designed to amplify the upstream and downstream homology arms of the target gene, wherein the forward primer of the upstream homology arm contains the upstream homology arm sequence of the pJOE-cas12f1, and the reverse primer of the downstream homology arm contains the downstream homology arm sequence of the pJOE-cas12f1. The upstream homology arm DNA fragment and the downstream homology arm DNA fragment are obtained by PCR amplification, and connected into a fusion fragment, namely Donor DNA, and then the Donor DNA is fused with the previously constructed pJOE-sgRNA12-cas12f1 plasmid to finally obtain the CRISPR / Cas12f1 plasmid;

[0010] Step 2: Bacillus subtilis genome editing

[0011] Transform the CRISPR / Cas12f1 plasmid into Bacillus subtilis competent cells and discard the plasmid to complete Bacillus subtilis genome editing;

[0012] Step 3: Verify the editing results

[0013] Design primers to amplify the target gene of the edited host and verify it by sequencing the PCR product.

[0014] The preferred technical solution is: it also includes the steps of: using sgRNA to obtain a modified CRISPR / Cas12f1 plasmid, then using the genomic wild-type target gene as a homologous template to repair double-strand breaks, and editing the target gene of the BS168 host genome. After the genome editing is completed, the host discards the plasmid, randomly selects multiple colonies for target gene amplification, and sends them for sequencing, compares the sequencing results, and records the number of successfully edited colonies to calculate the editing success rate.

[0015] The preferred technical solution is: in step 1, using the pJOE8999 plasmid as a template, designing a reverse primer, performing reverse PCR loop P, amplification, and performing gel recovery and purification after gel running verification to obtain the basic skeleton part except for the sgRNA scaffold and cas9 protein.

[0016] The preferred technical solution is: the PCR reaction system is as follows: 2x PrimeSTAR Max Premix, 12.5ul; Primer1, 1ul; Primer2, 1ul; Template, 100ng; distilled sterile water up to 25ul.

[0017] The preferred technical scheme is: in step 2, first, use primers arm-up-dhbB-F and up-dhbB-arm-R to perform PCR on the upstream homologous arm of dhbB, and primers arm-down-dhbB-R and down-dhbB-arm-F to perform PCR on the downstream homologous arm to obtain pure upstream homologous arm DNA fragments and downstream homologous arm DNA fragments; then, use XbaI and SalI restriction endonucleases to cut the plasmid pjoe-dhbBgRNA-cas12f1, run the gel, and obtain a linear plasmid after gel recovery and purification, and connect the upstream homologous arm DNA fragment and the downstream homologous arm DNA fragment by seamless cloning of the linearized plasmid to obtain pjoe-dhbBgRNA-cas12f1-homo-arm; transform DH5a, apply it on an LB plate containing kanamycin resistance, culture overnight, verify the correctness of colony PCR, extract the plasmid, and obtain the plasmid pjoe-dhbBgRNA-cas12f1-homo-arm.

[0018] The preferred technical scheme is: the constructed plasmid pjoe-dhbBgRNA-cas12f1 is transformed into Bacillus subtilis competent cells, which are spread on LB plates containing 50 μg / mL kanamycin and 0.4% mannose for culture, and the grown colonies are marked on the plate. After the culture is completed, the marked colonies are spotted on LB plates without antibiotics, cultured at 50°C for 16-18h, and the plasmid is discarded. Finally, the corresponding colonies are spotted on LB plates containing 50 μg / mL kanamycin for overnight culture to verify whether the plasmid is lost.

[0019] The preferred technical solution is: after confirming that the genome editing is complete and the host plasmid is completely lost, test-dhbB-F1 and test-dhbB-R2 are used as primers to amplify the dhbB gene of the edited host, and agarose gel electrophoresis is performed to verify the PCR results. After obtaining the target band, the PCR product is sequenced. If the result is correct, the host is named BS168-P1.

[0020] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0021] The present invention utilizes CRISPR-Cas12f technology to rationally transform the Bacillus subtilis BS168 strain, and improves the yield of MK-7 by knocking out the isochorismate lyase in the competitive pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Pjoe-Ascas12f.

[0023] Figure 2Cas12f-mediated knockout of the dhbB gene.

[0024] Figure 3 Vitamin K2 production comparison chart.

[0025] Figure 4 Pictures of the morphology of engineered bacteria strains. DETAILED DESCRIPTION

[0026] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in the embodiments.

[0027] See also Figure 1-4 It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with the technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no technical substantive significance. Any modification of the structure, change of the proportion relationship or adjustment of the size. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention.

[0028] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0029] The technical solution of the present invention is further described in detail below with reference to examples.

[0030] Example 1: Construction of pJOE-cas12f1 plasmid expressing CRISPR / Cas12f1 system

[0031] 1. First, query the cas12f1 gene sequence of Sulfuric Acid Oxidants from the NCBI website, and then query the pCMV-AsCas12f1-HKRA-sgRNA_v7 plasmid from the addgene official website to determine the scaffolds sequence of sgRNA12. The codon-optimized Ascas12f1 gene sequence, scaffolds-Ascas12f1 was synthesized by General Biotech.

[0032] Using pJOE8999 plasmid as template, reverse PCR amplification was performed by designing reverse primers.

[0033] The reaction system is as follows:

[0034]

[0035]

[0036] The reaction conditions are as follows:

[0037] step Temperature / degrees time Step 1 98 3min Step 2 98 10s Step 3 55 5s Step 4 72 5s / kb Step 5 72 5min Step 6 4

[0038] Agarose gel electrophoresis was performed, and the F1-pJOE8999 and R2-pJOE8999 primers and pJOE8999 were used as templates for PCR. The PCR reaction solution was aspirated for PCR verification, and the PCR product with the correct PCR identification band was purified by gel recovery. Specifically, the Thermo Fisher gel recovery and purification kit / Thermo Scientific GeneJET gel recovery kit was used to obtain the linearized basic skeleton of sgRNA9 scaffolds and cas9 protein, which was named pJOE. The restriction endonuclease cutting sites were determined to be AfiII and BsaI, and the primers were rationally designed. 20bp homology arms and restriction sites were added to the 5' end of the forward and reverse primers of sgRNA-scaffold-cas12f1. The synthesized sgRNA-scaffold-cas12f1 fragment was connected to pJOE by seamless cloning to obtain the pJOE-cas12f1 plasmid expressing the CRISPR / Cas12f1 system.

[0039] Reaction system

[0040] Components system Linearized vector X n insert fragments <![CDATA[Y1-Y n ul]]> 5xCE MultiS Buffer 4ul Exnase MultiS 2ul <![CDATA[ddH2O]]> to 20ul

[0041] X / Y is used to calculate the amount of vector and each insert fragment; the optimal amount of each fragment = [0.02 x the number of base pairs of the fragment] ng. Reaction conditions: 37 degrees, 30 minutes; drop to 4 degrees or immediately cool on ice.

[0042] Example 2

[0043] Construction of pJOE-cas12f1-sgRNA(dhbB)-homo-Arm shuttle plasmid, knockout of isochorismate lyase dhbB and construction of engineered bacteria.

[0044] The dhbB gene sequence was queried from the NCBI website to determine the position of the dhbB gene in the BS168 genome sequence. According to the known PAM: 5, -TTTG, a suitable 20bp sgRNA sequence was manually designed. The annealing system was: total volume 10μL, sterile water 6.5uL, oligo1 (100μm) 1μL, oligo2 (100μm) 1μL, 10X T4 connection buffer 1μL, T4 PNK 0.5μL. The annealing reaction procedure was: 37℃ reaction for 30min, 95℃ reaction for 5min, and then cooled to 20℃ at a rate of 0.1℃ / s. The cooling process took at least 45min to obtain the double-stranded DNA fragment of sgRNA, which was stored in a 4℃ refrigerator and prepared for connection with the restriction vector. At the same time, the dhbBsgRNA annealed into double-stranded DNA was mixed with the restriction-cut linear plasmid at a molar ratio of 3:1 for GoldenGate reaction. The reaction system was: T4 DNA ligase buffer 1.5 μL, 10*cutsmart 1.5 μL, BsaI 1 μL, T4 DNA ligase 1 μL, pjoe-cas12f1 100 ng, sgRNA (diluted 200 times) 2 μL, and dd water was added to make up to 15 μL.

[0045] Golden Gate reaction conditions:

[0046]

[0047] After the reaction is finished, take 10 μL of the assembled product and use it directly for transformation, including:

[0048] 1. Take out the competent cells (e.g. DH5α Competent cells, Vazyme#C502) from the -80℃ freezer and thaw them on ice.

[0049] 2. Add 100 ng of the recombinant product to the competent cells in a clean bench, flick the tube wall, mix well, and let stand on ice for 30 minutes.

[0050] 3. Adjust the water bath to 42°C, heat shock the competent cells in the water bath for 45 seconds, and immediately cool them on ice for 2-3 minutes.

[0051] 4. Add 900 μl of LB medium without antibiotics into the clean bench and shake the culture in a shaker at 37°C and 220 r / min for 1 h.

[0052] 5. Centrifuge at 5,000 rpm for 5 minutes, discard 900 μl of supernatant with a pipette, resuspend the bacteria, and spread the bacterial solution evenly on the plate containing the correct resistance using a sterile spreader.

[0053] 6. Invert and culture for 12-16 hours until a single colony grows. Transfer to E. coli DH5a competent medium, spread on kana resistance plates, culture conditions at 37 degrees, and culture for 12-16 hours. After a single colony grows, select the correct transformant to extract the plasmid and sequence it. The recombinant plasmid is named pJOE-cas12f1-sg-dhbB.

[0054] PCR primers were designed based on the 1000bp upstream and downstream homology arm nucleotide sequences of the knockout target dhbB, and the upstream and downstream homology arm gene fragments dhbB-Up / Down were amplified using the B. subtilis BS168 genome as a template, and purified and recovered using an agarose gel kit; the PCR reaction system was: total volume 25 μL, sterile water 12.5 μL, upstream primer 1 μL, downstream primer 1 μL, template 1 μL, Prime star DNA Polymerase 12.5 μL; the PCR reaction program was: 98°C 3 min, 98°C 10 s, 55°C 5 s, 72°C 5 s / kb, step 2-4 cycle number 35, 72°C 10 min, 4°C+∞.

[0055] PCR reaction conditions:

[0056]

[0057] 3. Perform agarose gel electrophoresis on the amplified upstream and downstream homology arm gene fragments dhbB-Up / Down to confirm the correctness of the PCR results and verify the correct use of the SanPrep column PCR product purification kit to purify and recover the gene fragments.

[0058] 4. The recombinant plasmid pJOE-cas12f1-sg-dhbB was digested with SalⅠ and XbaⅠ at 37℃ for 1h, and the digested product was purified and recovered using an agarose gel kit.

[0059] 5. The enzyme-digested product was connected with the purified dhbB-Up / Down gene fragment by seamless cloning and transformed into E.coli DH5a.

[0060] 6. Take 100 μL of freshly prepared E.co1i DH5a competent cells, add 10 μL of the ligation fragment, put it in an ice bath for 30 minutes, and put the centrifuge tube in 42℃ for 45 seconds. Then quickly put it in an ice bath for 2 minutes. Add 500 μL of LB liquid culture medium to the centrifuge tube, shake and culture at 37℃ for 1 hour. Centrifuge at 5000 rpm for 5 minutes, take 100 μL of culture solution and spread it on the kana resistance plate, and culture it at 37℃ for 12-16 hours. The obtained single colony was verified by colony PCR and sequencing, and the constructed recombinant plasmid was named pJ0E-dhbB.

[0061] 7. The successfully constructed plasmid pJ0E-dhbB was transformed into B. subtilis 168 by the Spizizen method. The constructed engineering strain was named B. subtilis 168-dhbB. The electrophoresis diagram of the successfully constructed strain is shown in the accompanying figure. Figure 2 shown.

[0062] 8. Elimination of plasmids:

[0063] The obtained B. subtilis 168 with dhbB knocked out was inoculated into a 5 ml LB test tube and cultured at 50°C, 200 rpm for 12 h. Single colonies were obtained by three-zone streaking. The single colonies were spotted onto an antibiotic-free LB solid plate and an LB solid plate containing 50 ug / m kanamycin, respectively, and cultured in a 37°C incubator for 12 h. The colonies that grew on the antibiotic-free plate but not on the Kana plate were selected and inoculated into a 5 mL LB test tube. After culturing for 12 h, the final knockout strain was obtained and named B. subtilis168-P1.

[0064] Embodiment 3:

[0065] Subtilis 168 and engineered strains shake flask fermentation:

[0066] (1) Bacteria activation:

[0067] The culture medium is LB solid medium, the ingredients are: peptone 10g / L, NaCl 10g / L, yeast powder 5g / L, agar powder 15g / L. Use an inoculation loop to take a loop of bacterial liquid from the glycerol tube, streak three zones on the antibiotic-free solid medium plate, and culture at 37℃ for 12h.

[0068] (2) Seed culture:

[0069] The components of the seed culture medium are: peptone 10 g / L, NaCl 10 g / L, yeast powder 5 g / L, agar powder 15 g / L, pH 7.0. A single colony was picked from the plate culture medium in step (1) and inoculated into a 250 ml Erlenmeyer flask containing 50 ml of seed culture medium, and cultured at 37 °C, 220 rpm for 12 h.

[0070] (3) Shake flask fermentation:

[0071] The fermentation medium components are: soy peptone 60g / L, sucrose 10g / L, glycerol 30mL / L, yeast extract 5g / L, MgSO4.7H2O 0.5g / L, K2HPO4 3g / L, and the rest is water, pH 7.0. The seed liquid was inoculated into 50mL of fermentation medium at a 10% inoculation rate, and cultured with shaking at 40°C and 220rpm for 168h. After HPLC detection, the WT vitamin K2 production can reach up to 30.8mg / L; the dhbB knockout strain has a production of 53.36mg / L, which is 20.2% higher than WT. Figure 3 shown.

[0072] Patented strains of engineered bacteria:

[0073] 1. Starting strain: Bacillus subtilis BS168.

[0074] Background information: Bacillus subtilis is a Gram-positive bacterium commonly used in industrial fermentation and genetic engineering research. Its excellent protein secretion ability and recognized safety make it an important industrial production host.

[0075] Characteristics: BS168 strain is a standard strain of Bacillus subtilis, widely used in scientific research and industrial applications. Its genome has been completely sequenced, providing a good basis for gene editing and functional research.

[0076] 2. Methods for constructing engineered strains

[0077] Gene editing technology: CRISPR / Cas12f gene editing technology was used. CRISPR / Cas12f is a new type of CRISPR system with a smaller protein size and efficient DNA cutting ability, suitable for a variety of gene editing applications.

[0078] Target gene: In the BS168 strain, the dhbB gene of the competitive pathway was knocked out. The dhbB gene is related to the bacterial metabolic pathway. Knocking out this gene can reduce metabolic competition and increase the yield of the target product.

[0079] 3. Characteristics and advantages of engineered strains

[0080] Metabolic optimization: By knocking out the dhbB gene, the engineered strain was optimized in the metabolic pathway, reducing unnecessary metabolic competition and potentially improving the synthesis efficiency of the target product.

[0081] Advantages of CRISPR / Cas12f:

[0082] High efficiency: The Cas12f system has efficient DNA cutting ability and is suitable for a variety of gene editing applications.

[0083] Compactness: The small size of the Cas12f protein facilitates manipulation and expression in the genome.

[0084] Application prospects: This engineered strain may show higher production efficiency in industrial fermentation and is suitable for the production of specific metabolites or enzyme preparations.

[0085] 4. Patent strain preservation information

[0086] Bacillus subtilis 168. Deposit date: January 9, 2025.

[0087] Depository: China Center for Type Culture Collection (CCTCC, Wuhan).

[0088] Deposit number: CCTCC AB 2025001.

[0089] Storage conditions: The engineered strains need to be stored at -80°C.

[0090] 5. Summary

[0091] The engineered strain used CRISPR / Cas12f technology to edit the gene of Bacillus subtilis BS168, knocking out the dhbB gene in the competitive pathway, thereby optimizing the metabolic pathway. It has potential application value in industrial production and scientific research. Specific strain preservation information needs to be queried and obtained according to the database of the preservation institution.

[0092] The primers used in the patent are as follows:

[0093]

[0094] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A method for editing the genome of Bacillus subtilis based on the CRISPR-Ascas12f system, characterized in that: The following steps are involved: Step 1: Construction of CRISPR / Cas12f1 plasmid Using pjoe8999 plasmid as template, reverse PCR was performed to obtain a backbone plasmid retaining the replicon and terminator; and pJOE-cas12f1 was formed by seamless cloning. sgRNA-F and sgRNA-R were annealed and connected to pJOE-cas12f1 via GoldenGate to obtain pJOE-sgRNA12-cas12f1 plasmid; Primers are designed to amplify the upstream and downstream homology arms of the target gene, wherein the forward primer of the upstream homology arm contains the upstream homology arm sequence of the pJOE-cas12f1, and the reverse primer of the downstream homology arm contains the downstream homology arm sequence of the pJOE-cas12f1. The upstream homology arm DNA fragment and the downstream homology arm DNA fragment are obtained by PCR amplification, and connected into a fusion fragment, namely Donor DNA, and then the Donor DNA is fused with the previously constructed pJOE-sgRNA12-cas12f1 plasmid to finally obtain the CRISPR / Cas12f1 plasmid; Step 2: Bacillus subtilis genome editing Transform the CRISPR / Cas12f1 plasmid into Bacillus subtilis competent cells and discard the plasmid to complete Bacillus subtilis genome editing; Step 3: Verify the editing results Design primers to amplify the target gene of the edited host and verify it by sequencing the PCR product.

2. The method for editing the Bacillus subtilis genome based on the CRISPR-Ascas12f system according to claim 1, characterized in that: The method also includes the following steps: using sgRNA to obtain a modified CRISPR / Cas12f1 plasmid, and then using the wild-type target gene in the genome as a homologous template to repair double-strand breaks, editing the target gene in the BS168 host genome. After genome editing is completed, the host discards the plasmid, randomly selects multiple colonies for target gene amplification, and sends them for sequencing, compares the sequencing results, and records the number of successfully edited colonies to calculate the editing success rate.

3. The method for editing the Bacillus subtilis genome based on the CRISPR-Ascas12f system according to claim 1, characterized in that: In step 1, using the pJOE8999 plasmid as a template, reverse primers were designed to perform reverse PCR loop P, amplification, and gel recovery and purification were performed after gel running verification to obtain the basic skeleton parts except for the sgRNA scaffold and cas9 protein.

4. The method for editing the Bacillus subtilis genome based on the CRISPR-Ascas12f system according to claim 3, characterized in that: The PCR reaction system is as follows: 2x PrimeSTAR Max Premix, 12.5ul; Primer1, 1ul; Primer2, 1ul; Template, 100ng; distilled sterile water up to 25ul.

5. The method for editing the Bacillus subtilis genome based on the CRISPR-Ascas12f system according to claim 1, characterized in that: In step 2, first, primers arm-up-dhbB-F and up-dhbB-arm-R are used to perform PCR on the upstream homology arm of dhbB, and primers arm-down-dhbB-R and down-dhbB-arm-F are used to perform PCR on the downstream homology arm to obtain pure upstream homology arm DNA fragments and downstream homology arm DNA fragments; then, the plasmid pjoe-dhbBgRNA-cas12f1 is cut using XbaI and SalI restriction endonucleases, gel run, gel recovery and purification are performed to obtain a linear plasmid, and the upstream homology arm DNA fragment, the downstream homology arm DNA fragment, and the linearized plasmid are seamlessly cloned and connected to obtain pjoe-dhbBgRNA-cas12f1-homo-arm; DH5a is transformed, coated on an LB plate containing kanamycin resistance, cultured overnight, colony PCR is verified to be correct, and plasmid extraction is performed to obtain the plasmid pjoe-dhbBgRNA-cas12f1-homo-arm.

6. The method for editing the Bacillus subtilis genome based on the CRISPR-Ascas12f system according to claim 1, characterized in that: The constructed plasmid pjoe-dhbBgRNA-cas12f1 was transformed into Bacillus subtilis competent cells, spread on LB plates containing 50 μg / mL kanamycin and 0.4% mannose for culture, and the grown colonies were marked on the plates. After the culture was completed, the marked colonies were spotted on LB plates without antibiotics and cultured at 50°C for 16-18h, the plasmid was discarded, and finally the corresponding colonies were spotted on LB plates containing 50 μg / mL kanamycin for overnight culture to verify whether the plasmid was lost.

7. The method for editing the Bacillus subtilis genome based on the CRISPR-Ascas12f system according to claim 1, characterized in that: After confirming that the genome editing was complete and the host plasmid was completely lost, test-dhbB-F1 and test-dhbB-R2 were used as primers to amplify the dhbB gene of the edited host, and agarose gel electrophoresis was performed to verify the PCR results. After obtaining the target band, the PCR product was sequenced. If the result was correct, the host was named BS168-P1.

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